基于尼罗红染色分析金藻总脂动态积累

382Vol.38,No.220143ACTAHYDROBIOLOGICASINICAMar.,2014收稿日期:2013-01-21;修订日期:2013-12-16基金项目:(200805039);(GHME2001SW02);(41076108,41106148,30870247);(2011GHY11533)作者简介:(1987—),,;;E-mail:[e-mail]通信作者:,,;[e-mail]:10.7541/2014.45周文俊1郑立1韩笑天2郑明刚1张魁英1高伟1崔志松1(1.,266061;2.,266060):Isochrysissp.CCMM5001,:,;1323µmol/L148.0µmol/(m2·s)25℃;441µmol/L92.5µmol/(m2·s)15℃;,63.3%22mg/(L·d):;;;;中图分类号:Q949.2;Q-33文献标识码:A文章编号:1000-3207(2014)02-0312-08,[1—3],,[4],,[4—6],Chlorellaproto-thecoides[7]Oedogoniumsp.[8]Chlorellazofingienesis[9]Haematococcuspluvialis[10]Dunaliellatertiolecta[11],,,[12],,,,,,,,[13—16],450—500nm528nm,(Fluorescenceinten-sity,FI)()[17—19],,,,,,,2:313,Isochrysissp.CCMM5001,40%,C14-C1880%,,,,,,,11.1Isochrysissp.CCMM5001,f/2[20],92.5µmol/(m2·s),16h︰8h,(25±1)℃1.2(TecanInfiniteM200)(NikonECLIPSETE2000-U)-(AgilentGC7890A-MS5975C)(NileRed)J&KScientific,0.1g/L,–20℃1.3,1mL1×106cells/mL,0.01mL1mg/L,,20℃10min,();96,200µL,480nm580nm,,3,31.1,,,0.1g-(%)()1,1.110%f/2,,表1金藻Isochrysissp.CCMM5001的不同培养条件Tab.1DifferentcultureconditionsforIsochrysissp.CCMM5001CultureconditionNconcentration(µmol/L)Illuminationintensity[µmol/(m2·s)]Temperature()℃Nexperiment4418821323176492.525Illuminationexperiment88237.092.5148.025Temperatureexperiment88292.5152535100mL,0.45µm,,-,-,HP-5MS(30m×0.25mm×0.25µm):,1mL/min,280,℃280;℃:50,℃2min,6/min℃300,℃15min;,1µL:EI,1200V,230,℃150,Scan℃1.43,(Mean±SD)OriginPro7.53143822.1-(1),,,,0.5µm,,1µm,1Isochrysissp.CCMM5001Fig.1TheresultoffluorescencemicroscopeexaminationofIsochrysissp.CCMM5001stainedwithNileRed,,,-,,2(R2=0.9572),,2.2,2.1,,≤1323µmol/L,,2(R2=0.9572)Fig.2ThelinearrelationshipbetweentheFIofsinglecellandcontentoftotallipids(R2=0.9572)1323µmol/L11d2.7×107cells/mL;1764µmol/L,(3A),441µmol/L1764µmol/L,,13d882µmol/L51.2%;,,,(3B),[37µmol/(m2·s)][148µmol/(m2·s)],,,2.6×107cells/mL(4A),(4B)(15)℃(35)℃,,25℃2.4×107cells/mL(5A),15℃,7d50.8%(5B),,,,2:3153Isochrysissp.CCMM5001(A)(B)Fig.3TheinfluenceofdifferentNconcentrationsongrowthcurves(A)andlipidsaccumulation(B)ofIsochrysissp.CCMM50012.32.2,1323µmol/L,,;92.5µmol/(m2·s),1×107cells/mL;25℃,,15℃,,25℃15℃5—7d,,(2)(6),[1323µmol/L,92.5µmol/(m2·s),25]℃,:,,2.5×107cells/mL;,,11d63.3%,44.2%(7),:882µmol/L,;,≥92.5µmol/(m2·s);25℃,;,15mg/(L·d),4Isochrysissp.CCMM5001(A)(B)Fig.4Theinfluenceofdifferentilluminationintensitiesongrowthcurves(A)andlipidsaccumulation(B)ofIsochrysissp.CCMM5001316385Isochrysissp.CCMM5001(A)(B)Fig.5Theinfluenceofdifferenttemperaturesongrowthcurves(A)andlipidsaccumulation(B)ofIsochrysissp.CCMM50016Isochrysissp.CCMM5001Fig.6ThecomparisonofinfluenceongrowthcurvesandlipidsaccumulationofIsochrysissp.CCMM5001betweencommoncul-tivationandtwo-stagecultivation45%,22mg/(L·d),GC-MS(3),,C14:0C16:0,C18:1,C14-C18,88%,,表2金藻Isochrysissp.CCMM5001两阶段培养法的培养条件Tab.2Thecultureconditionsoftwo-stagecultivationforIsochrysissp.CCMM5001Culturecondition(7d)Stageone(culturefor7days)(6d)Stagetwo(culturefor6days)Nconcentration(µmol/L)13231323Illuminationintensity(µmol/(m2·s))92.5148.0Temperature()℃251533.1,,AlonzoMayzaud[13],[17]Chen,etal.,0.25—2mg/L,,10min[21],(0—1.075×107cells/mL)(R2=0.9912),1.075×107cells/mL(8),,,Chen,etal.(Chaetoceroslauderi)(Emilianiahuxleyi)(Crypthecodiniumcohnii)2:3177Isochrysissp.CCMM5001Fig.7TheproductionrateoftotallipidsofIsochrysissp.CCMM5001underdifferentcultureconditions表3普通培养法和两阶段培养法对金藻Isochrysissp.CCMM5001主要脂肪酸组成的影响对比Tab.3ThecomparisonofinfluenceonfattyacidcompositionofIsochrysissp.CCMM5001betweencommoncultivationandtwo-stagecultivation(%)FattyacidcompositionTwo-stagecultivationCommoncultivationC14:023.5±0.828.0±0.9C16:15.8±0.24.1±0.1C16:020.0±0.723.2±0.9C18:23.9±0.12.9±0.1C18:135.2±1.330.8±1.2C20:50.2±0.1—C22:610.6±0.311.0±0.3UFA55.6±2.148.8±1.9FA44.4±1.751.2±1.9PUFA14.6±0.513.9±0.4C14-C1888.3±3.189.0±3.38Isochrysissp.CCMM5001Fig.8TheeffectofcelldensityonthefluorescenceintensityoftheIsochrysissp.CCMM5001stainedwithNilereddye(Nannochloropsissp.)(Chlorellasp.),DMSO,25%DMSO[21],[17—19,22],,,,(0.2g),,,963.2,,,[23,24];,N/P,,,,,,,,Isochrysissp.CCMM500125℃[23],,,[25][26],[148µmol/(m2·s)],31838,,:,[27—29],Isoch-rysissp.CCMM500115℃,,Isochrysissp.CCMM5001,,4Isochrysissp.CCMM5001,,Isochrysissp.CCMM5001:1323µmol/L148.0µmol/(m2·s)25℃441µmol/L92.5µmol/(m2·s)15℃,63.3%22mg/(L·d)[1]FelizardoP,CorreiaMJN,RaposoI,etal.Productionofbiodieselfromwastefryingoils[J].WasteManagement,2006,26(5):487—494[2]KulkarniMG,DalaiAK.Wastecookingoil-aneconomicalsourceforbiodiesel:Areview[J].Industrial&EngineeringChemistryResearch,2006,45(9):2901—2913[3]SongDH,FuJJ,ShiDJ.Exploitationofoil-bearingmicroalgaeforbiodiesel[J].ChineseJournalofBiotechnology,2008,24(3):341—348[4]SchenkPM,Thomas-HallSR,StephensE,etal.Secondgenerationbiofuels:high-efficiencymicroalgaeforbiodieselproduction[J].BioenergyResearch,2008,1(1):20—43[5]ChistiY.Biodieselfrommicroalgae[J].BiotechnologyAdvances,2007,25(3):294—306[6]HuQ,SommerfeldM,JarvisE,etal.Microalgaltriacylglycerolsasfeedstocksforbiofuelproduction:perspectivesandadvances[J].ThePlantJournal,2008,54(4):621—639[7]MiaoXL,WuQY.Biodieselproductionfro